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22 Nanotechnology andBiomaterials

J. Brock Thomas and Nicholas A. Peppas University of Texas, Austin, Texas

Michiko Sato and Thomas J. WebsterPurdue University, West Lafayette, Indiana

CONTENTS

22.1 Introduction 22.2 Nanotechnology in Biomaterials Science22.3 Current Research Efforts to Improve Biomedical Performance at the Nanoscale22.4 Soft Biomaterials

22.4.1 Structural Characteristics 22.4.2 Surface Properties 22.4.3 Biomimetics22.4.4 Nanoscale Biopolymer Carriers

22.5 Ceramic Nanomaterials 22.5.1 Increased Osteoblast Functions 22.5.2 Increased Osteoclast Functions 22.5.3 Decreased Competitive Cell Functions22.5.4 Increased Osteoblast Functions on Nanofibrous Materials

22.6 Metal Nanomaterials 22.7 Polymeric Nanomaterials22.8 Composite Nanomaterials 22.9 Areas of Application

22.9.1 Drug Delivery 22.9.2 Tissue Engineering 22.9.3 Biological Micro-Electro-Mechanical Systems

22.10 Considerations and Future DirectionsAcknowledgments References

22.1 INTRODUCTION

Biomaterials have received a considerable amount of attention over the last 30 years as a means of treat-ing diseases and easing suffering. The focus of treatment is no longer a conventional pharmaceuticalformulation but rather a combination of device-integrated biomaterial and the necessary therapeutic

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Copyright 2006 by Taylor & Francis Group, LLC

treatment. Biomaterials have found applications in approximately 8000 different kinds of medicaldevices [1], which have been used in repairing skeletal systems, returning cardiovascular functionality,replacing organs, and repairing or returning senses [2]. Even though biomaterials have had a pro-nounced impact in medical treatment, a need still exists to be able to design and develop better poly-mer, ceramic, and metal systems [3].

Polymeric biomaterials originated as off-the-shelf materials that clinicians were able to use insolving a problem, for example, dialysis tubing was originally made of cellulose acetate, vasculargrafts were fabricated from Dacron, and artificial hearts were molded from polyurethanes (PUs) [2].However, these materials did not possess the chemical, physical, and biological properties neces-sary to prevent further complications. Recent advances in synthetic techniques have allowed theseproperties to be imparted on polymeric biomaterials, which help to alleviate accompanying bio-compatibility issues. Nanotechnology, as this chapter will describe, further adds to the ability ofchemically tailoring polymeric materials to provide more opportunities for revolutionary break-throughs in the science and technology associated with developing novel devices. Undoubtedly,nanoscale science and engineering has the potential to have a profound impact on medical scienceand technology, which will lead to improved diagnostics and enhanced therapeutic methods [4].

For ceramics and metals, similar advancements through the use of nanotechnology can be envi-sioned. For example, bioimplants, administered to humans over the past nine decades, have mostlybeen synthetic prostheses consisting of ceramic or metal particles and grain sizes with conventionaldimensions (of the order of 1 to 104 µm). But the lack of sufficient bonding of these syntheticimplants to surrounding body tissues has, in recent years, led to the investigation of novel materialformulations. One such category of materials, nanophase ceramics and metals (or materials withconstituent components less than 100 nm in at least one direction), can be used to synthesizeimplants with surface roughness similar to that of natural tissues. Natural tissues have numerousnanometer features available for cellular interactions since they are composed of many nanostruc-tures (specifically, proteins). Several nanophase ceramic and metal biomedical implants are cur-rently being investigated, and are likely to gain approvals for clinical use in the near future.

One area in which nanophase polymers, ceramics, and metals are being heavily investigatedinvolves orthopedic/dental applications and this is for a good reason. Since 1990, the total number ofhip replacements, which is the replacement of the femoral and hip bones, has been steadily increasing[5–11]. In fact, in the United States alone in 2000 there were 152,000 total hip replacements — a 33%increase from the number performed in 1990 and a little over half of the projected number of total hipreplacements (272,000) by the year 2030 [5–8]. However, in 1997, 12.8% of the total hip arthroplas-ties were simply due to revision surgeries of previously implanted failed hip replacements [5–8].

The fact that such a high percentage of hip replacements performed every year are revision sur-geries is not surprising when you consider the life expectancy of the implant versus that of thepatient receiving the implant. Consistently, over 30% of those requiring total hip replacements havebeen below the age of 65 and even those at the age of 65 have a life expectancy of 17.9 years [9–11].Females, who comprise a majority of those receiving total hip replacements, have a life expectancyof 19.2 years at the age of 65. Since the longevity of implants ranges only from about 12 to 15 years,even the majority of those who receive bone implants at the age of 65 will require at least one revi-sion surgery before the end of their lives [9–11].

For the dental community the story is not any better. Since dental implants may be necessaryfor the young and old alike, it is imperative that they are able to last for the duration of the patient’slife. Recent studies have found that dental implants which have been used in over 300,000 cases inthe United States, have up to a 96% success rate (meaning that the implant was not mobile and wasnoninflamed) after 5 years, 80% after 10 years, and less than 75% after 15 years [5–11].

The above information strongly suggests that the longevity of the current prostheses is a reoc-curring problem for the orthopedic/dental community that has to be dealt with since currentapproaches clearly fail. Orthopedic implant failure can be due to numerous reasons, including poorinitial bonding of the implant to juxtaposed bone, generation of wear debris that lodges between the

606 Nanomaterials Handbook

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implant and surrounding bone to cause bone cell death, and stress and strain imbalances betweenthe implant and surrounding bone causing implant loosening and eventual failure [12]. Althoughthere are many reasons why implants fail, a central one is the lack of sufficient bone regenerationaround the implant immediately after insertion [12]. Shockingly, about a quarter of dental implantfailures (of those that failed between 3 and 6 months) are attributed to incomplete healing of theimplant to juxtaposed bone [5–11]. Importantly, this leads to eventual implant loosening andregions for possible wear debris to situate between the implant and surrounding bone, further com-plicating bone loss [12–15].

Nanotechnology is playing an important role in decreasing this failure. This is because, in orderto improve biomaterial performance and hence extend the lifetime of bone implants, it is essentialto design surface characteristics that interface optimally with select proteins and subsequently withpertinent bone cell types. That is, immediately after implantation, proteins will adsorb from plasmato biomaterial surfaces to control cell attachment and eventual tissue regeneration (Figure 22.1)[16,17]. Initial protein interactions that mediate cell function depend on many biomaterial proper-ties, including chemistry, charge, wettability, and topography [16,17]. Of significant influence forprotein interactions is surface roughness and energy [18–22], and this represents the promise ofnanophase materials in bone implant applications.

The critical factor for the merging of nanotechnology with medicine is the increasingly docu-mented, special, biologically improved material properties of nanophase implants compared to con-ventional formulations of the same material chemistry. This chapter will highlight a novel property ofnanophase materials that makes them attractive for use as implants: increased tissue regeneration. Workis ongoing in the domains of orthopedic, dental, bladder, neurological, vascular, cartilage, and cardio-vascular applications. However, only orthopedic applications, which are the closest to clinical applica-tions, will be emphasized here. This contribution will briefly articulate the seeming revolutionarychanges and the potential gains nanostructured materials can make for bone implant technology.

Nanotechnology and Biomaterials 607

Cytoskeleton

Integrin

Integrin receptors

Cell

Surface properties affecting protein interactions: Chemistry, wettability, topography, etc.Substrate

RGD

Ca2−�

�

Proteins(e.g., vitronectin, fibronectin, etc.)

Adhesive peptide sequence of proteins recognized by integrins(e.g., arginine-glycine-aspartic acid (RGD))

Cell membrane

FIGURE 22.1 Cell recognition of biomaterial surfaces controlled by initial protein interactions. Initial proteininteractions can influence cell adhesion and, thus, degree of bone tissue formation on biomaterials. Changing mate-rial properties will alter protein interactions and influence subsequent cell function. (Adapted and redrawn fromSchakenraad, J.M., in Biomaterials Science: An Introduction to Materials in Medicine, Ratner, B.D., Hoffman,A.S., Schoen, F.S., and Lemmons, J.E., Eds., Academic Press, New York, 1996, pp. 133–140. With permission.)

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22.2 NANOTECHNOLOGY IN BIOMATERIALS SCIENCE

The emergence of micro- and nanoscale science and engineering has provided new avenues forengineering materials with macromolecular and even down to molecular-scale precision, leading todiagnostic and therapeutic technologies that will revolutionize the way health care is administered.Biomaterials have evolved from off-the-shelf products (e.g., Dacron for vascular grafts) to materi-als that have been designed with molecular precision to exhibit the desired properties for a specificapplication, often mimicking biological systems [2,3].

Controlling interactions at the level of natural building blocks, from proteins to cells, facilitatesthe novel exploration, manipulation, and application of living systems and biological phenomena.Nanostructured tissue scaffolds and biomaterials are being applied for improved tissue design, recon-struction, and reparative medicine [23–26]. Nano- and micro-arrays have been established as the pre-ferred method for carrying out genetic and other biological (e.g., drug discovery) analysis on a massivescale [27]. Natural nanopores [28,29] and synthetic nanopores of tailored dimensions [30,31] are prob-ing, characterizing, and sequencing biological macromolecules and have demonstrated the possibilityto analyze the structure of individual macromolecules faster and cheaper [32]. Self-assembly is beingapplied to create new biomaterials with well-ordered structures at the nanoscale, such as nanofiberpeptide and protein scaffolds [33]. In addition, polymer networks with precisely engineered bindingsites have been created via molecular imprinting, where functional monomers are preassembled witha target molecule and then the structure is locked with network formation [34].

In medical diagnostics, the speed and precision with which a condition is detected directly impactsthe prognosis of a patient. Point-of-care (POC) diagnostic devices, which enable diagnostic testing(in vivo or ex vivo) at the site of care, can enhance patient outcomes by substantially abbreviated analy-sis times as a result of the intrinsic advantages of the miniature device and by eliminating the need forsample transport to an on-site or off-site laboratory for testing. The development of micro or minia-turized total analysis systems (µ-TAS), also referred to as lab-on-a-chip devices, has profoundlyimpacted the corresponding development of POC diagnostic devices. These µ-TAS devices integratemicrovalves, micropumps, micro-separations, microsensors, and other components to create miniaturesystems capable of analysis that typically requires an entire laboratory of instruments. Since beingintroduced as a novel concept for chemical sensing devices [35], µ-TAS devices have been applied asinnovative biological devices [36] and POC diagnostic devices [37,38]. With the further developmentof micro- and nanosensors, POC diagnostic devices will provide for improved medical management,leading eventually to self-regulated POC diagnostic devices that intermittently or continuously moni-tor the biological molecule of interest and deliver the therapeutic agents as required.

Additionally, nanoscale science and engineering have accelerated the development of noveldrug delivery systems and led to enhanced control over how a given pharmaceutical is administered,helping biological potential to be transformed into medical reality [39]. Micro- and nanoscaledevices have been fabricated using integrated circuit processing techniques and have been demon-strated to allow for strict control over the temporal release of the drug. Silicon microchips that canprovide controlled release of single or multiple chemical substances on demand via electrochemi-cal dissolution of the thin anode membranes covering microreservoirs have been created [40]. Theadvantages of this microdevice are that it has a simple release mechanism, very accurate dosing,ability to have complex release patterns, potential for local delivery, and possible biological drugstability enhancement by storing in a microvolume that can be precisely controlled. More recently,multi-pulse drug delivery from a resorbable polymeric microchip device was demonstrated [41].

In particular, the development of polymer systems that are able to interact with their environmentin an “intelligent” manner has led to novel materials and applications. These intelligent materials areattractive options as functional components in micro- and nanodevices, due to the ease with whichtheir recognition and actuation properties can be precisely tailored. In this section, neutral and intelli-gent polymers and networks based on environmentally responsive hydrogels and bio-mimetic poly-mer networks will be discussed for application as sensing/recognition elements in novel diagnostic


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devices, such as microsensors and microarrays, and therapeutic devices, for tailoring loading andrelease properties.

In addition to advances in polymer nanotechnology for sensing and recognizing changes inmicro-environments, advances have been made concerning tissue regeneration on ceramic and metal-lic nanomaterials. Broadly speaking, nanotechnology embraces a system whose core of materials isin the range of nanometers (10−9 m) [42–52]. The application of nanomaterials for medical diagno-sis, treatment of failing organ systems, or prevention and cure of human diseases can generally bereferred to as nanomedicine [51,52]. The branch of nanomedicine devoted to the development ofbiodegradable or nonbiodegradable prostheses fall within the purview of nanobiomedical scienceand engineering [51,52]. Although various definitions are attached to the word “nanomaterial” bydifferent experts, the commonly accepted concept refers nanomaterials as that material with the basicstructural unit in the range 1 to 100 nm (nanostructured), crystalline solids with grain sizes 1 to100 nm (nanocrystals), individual layer or multilayer surface coatings in the range 1 to 100 nm(nanocoatings), extremely fine powders with an average particle size in the range 1 to 100 nm(nanopowders) and, fibers with a diameter in the range 1 to 100 nm (nanofibers) [42,43].

Since nature itself exists in the nanometer regime, especially tissues in the human body [53], itis clear that nanotechnology can play an integral role in tissue regeneration. Specifically, bone iscomposed of numerous nanostructures — like collagen and hydroxyapatite (HA) that, most impor-tantly, provide a unique nanostructure for protein and bone cell interactions in the body (Figure 22.2)[50]. Although the ability to mimic constituent components of bone is novel in itself, there are addi-tional reasons to consider nanomaterials for tissue regeneration such as in orthopedic applications:their special surface properties compared to conventional (or micron constituent component struc-tured) materials [47–50]. For example, a nanomaterial has increased numbers of atoms at the surface,grain boundaries or material defects at the surface, surface area, and altered electron distributionscompared to conventional materials (Figure 22.3) [50]. In summary, nanophase material surfaces aremore reactive than their conventional counterparts. In this light, it is clear that proteins which influ-ence cell interactions that lead to tissue regeneration will be quite different on nanophase comparedwith conventional implant surfaces (Figure 22.1).


Cancellous bone

Cortical bone

Osteon

Microstructure

Macrostructure Sub-microstructure

Nanostructure

Sub-nanostructure

10−500 µm3−7 µm

0.5 µm

1 nm

Lamella Collagenfiber

Collagenfibril

Collagenmolecule

BonecrystalsHaversian

canal

FIGURE 22.2 Nanocomponents of bone provide a high degree of nanostructured surface roughness for bonecells. (Adapted and redrawn from Cowin, R., Handbook of Bioengineering, McGraw-Hill, New York, 1987.With permission.)

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Despite this, the evolution of tissue engineering has centered on the use of materials with non-biologically inspired micron surface features [55,56], mostly changing in chemistry or micron rough-ness but not degree of nanometer roughness (Figure 22.4). In this context, it should not be surprisingwhy the optimal tissue engineering material (in particular, to regenerate bone) has not been found.

22.3 CURRENT RESEARCH EFFORTS TO IMPROVE BIOMEDICAL PERFORMANCE AT THE NANOSCALE

Nanoscale materials currently being investigated for bone tissue engineering applications canbe placed in the following categories: ceramics, metals, polymers, and composites thereof.Each type of material has distinct properties that can be advantageous for specific bone regrowthapplications. For example, HA, a ceramic mineral present in bone (Figure 22.2), can also be madesynthetically. Ceramics, though, are not mechanically tough enough to be used in bulk for large-scalebone fractures. However, they have found applications for a long time as bioactive coatings due totheir ionic bonding mechanisms favorable for osteoblast (or bone-forming cells) function [57].


Ti (Medical grade 2) at amagnification of ×100

Ti (Medical grade 2) at amagnification of ×400

FIGURE 22.4 Conventional grain size of currently used orthopedic implants. Bar = 10 and 1 µm for the leftand right micrograph, respectively.

Altered electrondelocalizationNanophase

material

Num

ber

(arb

itrar

y un

its)

Conventionalmaterial

Bulk Surface

OH−

OH−

OH−

OH−OH

−OH

−OH

−OH

−OH

−

(a) Conventional(grain size: > 100 nm)

(b)

(b) Nanophase(grain size: < 100 nm)

(a)

FIGURE 22.3 Special surface properties of nanophase materials. (a) Higher number of atoms at the surfacefor nanophase compared to conventional materials. (b) Nanophase materials have higher surface areas, possessgreater numbers of material defects at the surface, and altered electron delocalization. Such special propertieswill influence protein interactions for controlling cell functions. (Adapted and redrawn from Klabunde, K.J.et al., J. Phys. Chem., 100, 12141, 1996. With permission.)

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Unlike ceramic materials, metals are not found in the body. Due to their mechanical strengthand relative inactivity with biological substances, metals (specifically, Ti, Ti6Al4V, and CoCrMo)have been the materials of choice for large bone fractures [55,56]. Polymers exhibit unique proper-ties (such as viscosity, malleability, moldability) and possess mechanical strength that is compara-ble with many soft (not hard) tissues in the body [54]. To date, because of their excellent frictionproperties, polymers (like ultra-high-molecular-weight polyethylene) have been primarily used asarticulating components of orthopedic joint replacements [58]. Additionally, some polymers (par-ticularly the polyester family) can be resorbed or degraded in the body, which opens the windowfor controllable repair of damaged bone that is actively being investigated in tissue engineering cir-cles. Lastly, composites of any or all of the above, can be synthesized to provide a wide range ofmaterial properties to increase bone implant performance [59]; such ability to tailor compositeproperties to specific orthopedic applications makes them attractive.

Owing to the numerous materials currently being used and investigated in orthopedics, thisreview will cover select efforts to create nanoscale surfaces in all of these categories: ceramics, met-als, polymers, and composites. Several current and potential materials that have shown promise innanotechnology for bone biomedical applications as well as needed future directions, will beemphasized.

22.4 SOFT BIOMATERIALS

Polymers are only one of the four major classes of biomaterials; however, polymeric biomaterialshave a significant advantage over metals, ceramics, and natural materials in that they can be chem-ically synthesized or modified according to the desired application. Hydrogels, hydrophilic, andcross-linked polymeric structures, have received considerable attention for use in biomedical appli-cations due to the biocompatible nature of their physical properties [60–62]. Poly(hydroxyethylmethacrylate) [63] is the most widely used hydrogel, and the cross-linked variant has been fre-quently used in soft contact lenses [64,65]. Hydrogels are also widely used in pharmaceutical appli-cations especially for the oral delivery of therapeutic proteins [66]. This class of materials is alsocapable of responding to changes in the external pH (Figure 22.5), temperature, ionic strength,nature and composition of the swelling agent, enzymatic or chemical reaction, and electrical ormagnetic stimuli [67].

Much consideration must be given when designing a material for a specific application. Certainproperties of the material must be controlled so as to perform the necessary function and elicit the


pH decrease

Mg, � decrease

�

�

FIGURE 22.5 Network structural changes due to variations on environmental pH. Higher pHs disrupt theinterpolymer complexes and ionic moieties deprotonize leading to extensive swelling (left). As pH isdecreased, interactions between the tethered grafts with the protonized ionic moieties increase leading to theformation of interpolymer complexes.

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appropriate response. These properties can be tailored to the specific need by carefully controllingstructural characteristics, modifying the surface properties, and employing biomimesis in the mate-rial design. Hydrogels are a suitable material for biomedical devices because their properties can becontrolled during or after the chemical synthesis, providing a significant amount of flexibility to aidin need-specific design applications.

22.4.1 STRUCTURAL CHARACTERISTICS

Hydrogel networks are prepared via chemical cross-linking, photopolymerization, or irradiativecross-linking [68] with the behavior of the materials dependent on their equilibrium and dynamicswelling behavior in water. Flory [69] was the first to develop the theory to explain the swellingbehavior of cross-linked polymers based upon a Gaussian distribution of the polymer chains.Various parameters have been employed to define the equilibrium-swelling behavior. The volumedegree of swelling, Q, is the ratio of the actual volume of a sample in the swollen state divided byits volume in the dry state and q, the weight degree of swelling, is the ratio of the weight of theswollen sample to that of the dry sample [62]. The basic structure of the hydrogel is described bythe molecular weight between cross-links, M�c, and the cross-linking density, ρx and can be calcu-lated from Equations (22.1) and (22.2), respectively. The structural characteristics influence thediffusion coefficient of solutes through the network, optical properties, mechanical properties, andsurface mobility:

� � (22.1)

ρx � (22.2)

These key properties of the hydrogel are typically determined during initial synthesis of the bio-material and are representative of the bulk structure of the material. Advances in nanotechnologyhave afforded the ability to refine further the structure by molecularly engineering the hydrogel toimpart a recognitive capacity. Domains within the molecularly designed hydrogel are able to rec-ognize specific molecules through highly select noncovalent interactions between the buildingblocks of both the hydrogel network and the recognizable molecule [70].

22.4.2 SURFACE PROPERTIES

In addition to the bulk structural characteristics of the biomaterial, the surface plays a key role inthe ability of the material to function as designed. A surface provides a low-energy barrier to mobil-ity, a high accessibility for reaction, enhanced reaction turnover rates, and ultimately allows formolecular recognition [71]. As the first biomaterials were developed (intraocular lenses, hip jointreplacements, and blood-contacting devices), the importance of the surface properties, protein–sur-face interaction, and surface modification was evident to researchers [72,73]. Baier et al. [74] intro-duced methods with the ability to determine the driving mechanisms behind bioreactions. Hoffman[75] demonstrated that materials could be engineered so as to provide the desired biologicalresponses necessary for effective biomaterial functionality.

In this light, poly(ethylene glycol) (PEG) has received considerable attention for its use at thebiomaterial–host interface to prevent protein fouling [76–78]. Castner and Ratner [71] list otherstrategies in addition to PEG that have been employed in biomaterials to prevent protein adsorption.However, possessing the ability to design surface properties has not been able to fully solve the issues

1�υ�M�c�

�Vυ�

1�[1n(1�υ2,S)�υ2,S�χυ 2

2,S]��

�υ1/32,S��

υ22,S��

2�M�c

1�M�c


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associated with the biocompatibility of the material. True biocompatibility will be achieved whenabsorbing proteins are able to maintain their conformation, eliminating the detrimental foreign bodyreaction to this newly formed unnatural layer of denatured proteins [71]. The surface of the materialmust be designed so as to provide components inherent to the natural wound healing process. Thisis an important contribution nanotechnology can make to improve biomaterial performance.

22.4.3 BIOMIMETICS

The design and development of biomaterials have primarily been focused on the biocompatibilityof the device with its surroundings, overlooking the biomolecular interactions that occur. The par-adigm for biomaterial characterization has shifted from one focusing on broad design parameters toone that focuses on eliciting specific molecular responses from the physiological environment.Biomimetic materials emulate nature and mimic biological architecture to elicit a desired cellularresponse [79,126]. Because of their hydrophilic structure, hydrogels inherently possess the charac-teristics of natural tissue; however, these materials in device form are much larger than individualcells. The major thrust of biomimesis is to design structures that have the ability to interact on asub-cellular basis.

In designing biomaterials one must understand the mechanisms by which cells interact for effi-cient biological mimicking. Three specific interactions that are crucial in material developmentinclude cell adhesion, morphogenic stimuli signaling, and endocytosis [79]; since all of these areevents controlled at the nanoscale, biomimetics is a critical field in nanotechnology.

22.4.4 NANOSCALE BIOPOLYMER CARRIERS

Polymer nanoparticle and nanosphere carriers (Figure 22.6 and Figure 22.7) are very attractive forbiomedical and pharmaceutical applications, due to their unique and tailorable properties. In thecase of polymer networks, the release profile can be precisely controlled through the design of itsmolecular structure, such as degree of cross-linking and ionic characteristics of the pendent func-tional groups [80,81].


FIGURE 22.6 TEM image of P(MAA-g-EG) nanospheres prepared from a MAA/EG molar feed ratio of 1:1.The P(MAA-g-EG) nanospheres were stained with uranyl acetate at pH 4.0.

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Polymer nanospheres have been molecularly designed to be responsive to the pH of their envi-ronment, enabling the protection of fragile therapeutic peptides (Figure 22.8) and proteins in theharsh, acidic stomach environment and then releasing them in the more amiable environment of theupper small intestine [82–84]. In addition, nanoparticle carriers have been designed to have stealthproperties, allowing extended residence time without being recognized by the immune system[85,86]. In other efforts, synthetic delivery systems, including polymeric nanoparticles, have beendeveloped for application in gene delivery [87,88]. By creating polymer drug delivery systems thatare biodegradable, the need for removal of the system postdelivery is eliminated, since the polymer


FIGURE 22.7 TEM image of P(MAA-g-EG) nanospheres prepared from a MAA/EG molar feed ratio of 1:1.The P(MAA-g-EG) nanospheres were stained with phosphotugstic acid pH 7.2.

15 µm

FIGURE 22.8 Optical section of a Caco-2 cell monolayer grown on microporous Transwell® plates obtainedwith a confocal microscope. FITC-labeled insulin (green) was added to the apical chamber of the cell mono-layer in the presence of poly(methacrylic acid-grafted-ethylene glycol) microparticles for 120 min, andimages were taken after fixing the cells with 3.7% formaldehyde.

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can be naturally resorbed by the body [89]. Also, a number of companies are reformulating insolu-ble drugs as nanoparticles and nanocrystals to control uptake through cellular membranes.

Microchips (Figure 22.9) have been created for the storage and then delivery of multiple drugsin a controlled manner. For instance, a solid-state silicon microchip that can provide controlledrelease of single or multiple chemical substances on demand was fabricated and demonstrated[40,90]. The release is achieved via electrochemical dissolution of the thin anode membranes cov-ering the micro-reservoirs filled with chemicals in solid, liquid, or gel form. The advantages of thismicrodevice include a simple release mechanism, very accurate dosing, ability to have complexrelease patterns, potential for local delivery, and possible biological drug stability enhancement bystoring in a micro-volume that can be precisely controlled. Recently, multi-pulse drug delivery froma resorbable polymeric microchip device was demonstrated [41].

The aforementioned microdevices demonstrate only a few examples of the wide variety ofnovel applications that exist for integration of micro- and nanofabrication technologies in drugdelivery, revealing the immaturity of the field. These novel drug delivery devices can enable effi-cient delivery that was unattainable with conventional drug delivery techniques, resulting in theenhancement of the therapeutic activity of a drug.

22.5 CERAMIC NANOMATERIALS

Perhaps slightly more mature, is the application of nanophase ceramics in bone tissue engineeringapplications. The next series of sections will highlight the improvement in bone regeneration thatcan be obtained through the use of ceramic nanotechnology.

22.5.1 INCREASED OSTEOBLAST FUNCTIONS

The first report correlating increased bone cell function with decreased material grain or particulatesize into the nanometer regime dates back to 1998 and involves ceramics [91]. Such reports describedhow in vitro osteoblast (bone-forming cell) adhesion, proliferation, differentiation (as measured byintracellular and extracellular matrix protein synthesis such as alkaline phosphatase), and calcium


(a)

50 µm

(b)

FIGURE 22.9 (a) 3-D projection of a micropatterned square array of a biomimetic polymer network basedon a cross-linked polyacrylamide obtained utilizing a confocal microscope. (b) A slice of the square array.

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deposition were enhanced on ceramics with particulate or grain sizes less than 100 nm [91–100].Specifically, this was first demonstrated for a wide range of ceramic chemistries including titania(Figure 22.10), alumina, and HA [93]. For example, four, three, and two times the amount of cal-cium-mineral deposition was observed when osteoblasts were cultured for up to 28 days onnanophase compared with conventional alumina, titania, and HA, respectively [95]. It is important tonote that for each respective nanophase and conventional ceramic mentioned in these first reports,similar chemistry and material phase were studied [91–100]. That is to say, only the degree ofnanometer surface features were altered between respective nanophase and conventional alumina,titania, and HA. This is important since as previously discussed it is well known that alterations insurface chemistry will influence bone cell function [12,55–59], but this was the first time changes inthe degree of nanometer roughness alone were reported to enhance bone cell responses [91].

Although these studies provided preliminary evidence that osteoblast functions can be pro-moted on nanostructured materials compared with conventional materials regardless of ceramicchemistry, Elias et al. [101] further described a study where the topography of compacted carbonnanometer fibers were transferred to poly-lactic-glycolic acid (PLGA) using well-establishedsilastic mold techniques. The same procedure was followed for compacts composed of conventionalcarbon fibers. The successful transfer of nanometer surface features in compacted carbon nanome-ter fibers and micron surface features in conventional fiber compacts were compared and is illus-trated in Figure 22.11 [101]. Importantly, osteoblast adhesion increased on PLGA molds made fromnanometer fibers compared to conventional carbon fibers [101]. Increased osteoblast functions werealso observed on the original nanometer fiber material compared with conventional carbon fibercompacts [101]. In this manner, this study provided further evidence of the importance of nanome-ter surface features (and not chemistry) in promoting functions of bone-forming cells.

Equally interesting, a step-function increase in osteoblast performance has been reported at distinctceramic grain sizes, specifically at alumina and titania spherical grain sizes below 60 nm [93]. This isintriguing since when creating alumina or titania ceramics with average grain sizes below 60 nm, adrastic increase in osteoblast function was observed compared to respective ceramics with grain sizesjust 10 nm higher (i.e., those with average grain sizes of 70 nm) [93]. This critical grain size for improv-ing osteoblast function is also of paramount importance since numerous other special properties (suchas mechanical, electrical, catalytic) of materials have been reported when grain size is specificallyreduced to below 100 nm [42–50]. With this information, evidence has been provided to show for thefirst time that the ability of nanophase ceramics to promote bone cell function is indeed limited to grainsizes (or subsequent surface features) below 100 nm, specifically below 60 nm [93]. Thus, anothernovel size-dependent property of nanostructured ceramics has been elucidated by these studies.


Microns

Microns

6

1.3

0

4

6

20

4

2

0

0

1.3

Microns

4

6

2

06

42

0Microns

(a) (b)

FIGURE 22.10 (a) Conventional and (b) nanophase titania. One of the first studies correlating increasedosteoblast function with decreasing ceramic grain size was carried out on titania as pictured here. (FromWebster, T.J. et al., Biomaterials, 20, 1221, 1999. With permission.)

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Although an exact explanation as to why greater bone regeneration is observed on smaller grainsize ceramics in the nanometer regime is not known to date, it is believed that the importance of thisspecific grain size in improving osteoblast function is connected with interactions of vitronectin (aprotein known to mediate osteoblast adhesion with linear dimensions remarkably similar to the crit-ical grain size of 60 nm mentioned above) [94,99]. Moreover, as mentioned previously, severalstudies have indicated that vitronectin and other proteins important for osteoblast adhesion are morewell spread and thus expose amino acid sequences to a greater extent when interacting withnanometer ceramics compared with conventional ceramics [94,99]. It is also intriguing to note thatnumerous investigators have confirmed that the minimum distance between protein ligands (such asarginine-glycine-aspartic acid or RGD) necessary for cell attachment and spreading is in thenanometer regime (specifically from 10 to 440 nm depending on whether the study was completedwith full proteins, protein fragments, or single RGD units) [102–107]. Therefore, an underlyingsubstrate surface that mediates protein spreading (as opposed to protein folding) to expose such lig-ands, coupled with a nanometer surface roughness to further project such ligands to the cell, maypromote cell adhesion due to this optimal ligand spacing.

22.5.2 INCREASED OSTEOCLAST FUNCTIONS

In addition to studies highlighting enhanced osteoblast function on nanophase ceramics, increasedfunctions of osteoclasts (bone-resorbing cells) have been reported on nanospherical compared withlarger grain size alumina, titania, and HA [100]. Specifically, osteoclast synthesis of tartrate-resist-ant acid phosphatase (TRAP) and subsequent formation of resorption pits was up to two timesgreater on nanophase compared to conventional ceramics such as HA. Coordinated functions ofosteoblasts and osteoclasts are imperative for the formation and maintenance of healthy new bone


PLGA mold from conventional carbon fibers

Unaltered PLGA PLGA mold from Nanophase carbon fibers

Nanophase carbonfiber compacts

Conventional carbonfiber compacts

FIGURE 22.11 Poly-lactic-glycolic acid (PLGA) molds of conventional and nanophase carbon fiber com-pacts. To highlight the importance of nanometer surface roughness regardless of substrate chemistry, studieshave shown increased functions of osteoblasts on PLGA molds of nanophase compacts compared to conven-tional carbon compacts. Studies have also shown increased functions of osteoblasts on compacts composed ofnanometer fibers compared to conventional carbon fibers. Bar = 1 µm. (From Elias, K.L. et al., Biomaterials,23, 3279–3287, 2002. With permission.)

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juxtaposed to an orthopedic implant [12]. Frequently, newly formed bone juxtaposed to implants isnot remodeled by osteoclasts and thus becomes unhealthy or necrotic [57]. At this time, the exactmechanism of greater functions of osteoclasts on nanophase ceramics is not known, but it may betied to the well-documented increased solubility properties of nanophase compared with conven-tional materials [48]. In other words, due to larger numbers of grain boundaries at the surface ofsmaller grain size materials, increased diffusion of chemicals (such as TRAP) may be occurring tosubsequently result in the formation of more resorption pits.

Collectively, results of promoted functions of osteoblasts coupled with greater functionsof osteoclasts imply increased formation and maintenance of healthy bone juxtaposed to an implantsurface composed of nanophase ceramics. In fact, although not compared with conventional grainsize apatite-coated metals, some studies have indeed demonstrated increased new bone formationon metals coated with nanophase apatite [108]. As shown in Figure 22.12, bone formation can beclearly seen on the surface of metals coated with nano-apatite, whereas there is no indication of newbone formation on the underlying metal without the coating [108]. Incidentally, coating metals withnanophase HA has been problematic [109]. For example, owing to their small grain size, techniques


Nano-apatite100–200 nmcrystal sizes

(a)

(b)

Nano-apatite-coated Ti

Morebonegrowth

Noncoated Ti

0.2 µm

FIGURE 22.12 Increased in vivo bone regeneration on titanium coated with nanophase apatite. Scanningelectron micrograph of nanometer-dimensioned apatite (specifically, between 100–200 nm in size) is depictedin (a). Increased bone regeneration in titanium cages when coated with nano-apatite is depicted in (b). (FromLi, P., J. Biomed. Mater. Res., 66, 79–85, 2003. With permission.)

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which use high temperatures (like plasma spray deposition) are not an option since they will resultin HA grain growth into the micron regime [109]. To circumvent such difficulties, some investiga-tors have allowed nanophase HA to precipitate on metal surfaces; this can be time consuming andnot very controllable [108]. In contrast, others have developed novel techniques which use high-pressure-based processes that do not significantly create elevated temperatures to coat nanophaseceramics on metals so as to retain their bioactive properties (Figure 22.13) [110].

22.5.3 DECREASED COMPETITIVE CELL FUNCTIONS

Importantly, it has also been shown that competitive cells do not respond in the same manner tonanophase materials as osteoblasts and osteoclasts do [94,101,111]. In fact, decreased functions offibroblasts (cells that contribute to fibrous encapsulation and callus formation events that may leadto implant loosening and failure [12] and of endothelial cells [cells that line the vasculature of thebody]) have been observed on nanophase compared with conventional ceramics [94]. In fact, theratio of osteoblast to fibroblast adhesion increased from 1:1 on conventional alumina to 3:1 onnanophase alumina [94].

Previously, such selectivity in bone cell function on materials has only been observed throughdelicate surface chemistry (e.g., through the immobilization of peptide sequences like Lys-Arg-Ser-Arg or KRSR) [112]. It has been argued that immobilized delicate surface chemistries may be compromised once implanted due to macromolecular interactions that render such epitopes non-functional in vivo. For these reasons, it is important to note that studies demonstrating selectenhanced osteoblast and osteoclast functions with decreased functions of competitive cells onnanophase materials have been conducted on surfaces that have not been chemically modified bythe immobilization of proteins, amino acids, peptides, or other entities [94,101,111]. Rather it is theunmodified, raw material surface that is specifically promoting bone cell functions.

Fibroblast function was also investigated in the same study that was previously mentioned inwhich Elias et al. transferred the topography of compacted carbon nanometer fibers compared to con-ventional fibers to PLGA using well-established silastic mold techniques (Figure 22.11) [102].Similar to the observed greater osteoblast adhesion already noted, decreased fibroblast adhesion wasmeasured on PLGA molds synthesized from carbon nanometer fibers compared to conventionalfibers [102]. Again, this was the same trend observed on the starting material of carbon nanometer


NanometerHA

FIGURE 22.13 Nanophase hydroxyapatite coated on titanium. Owing to elevated temperatures, traditionalcoating techniques, like plasma spray deposition, cannot be used to coat metals with nanophase ceramics. Thisprocess developed by Spire Biomedical (Bedford, MA) uses high pressure at low temperatures so as to notallow for grain growth. Bar = 1 µm (upper left).

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fiber compacts compared to conventional fiber compacts [102]. Thus, this study demonstrated theimportance of a nanometer surface roughness (and not chemical composition of the material) indecreasing functions of fibroblasts that may lead to undesirable fibrous encapsulation and callus for-mation events inhibiting osseointegration of orthopedic implants with surrounding bone.

22.5.4 INCREASED OSTEOBLAST FUNCTIONS ON NANOFIBROUS MATERIALS

Recently, researchers have further modified nanophase ceramics to simulate not only the nanome-ter dimension but also the aspect ratio of proteins and HA crystals found in the extra-cellular matrixof bone [111]. For example, consolidated substrates formulated from nanofibrous alumina (diame-ter � 2 nm, length � 50 nm; Figure 22.14) increased osteoblast functions in comparison with sim-ilar alumina substrates formulated from the aforementioned nanospherical particles [111].Specifically, Price et al. [111] determined a twofold increase in osteoblast cell adhesion density onnanofiber vs conventional nanospherical alumina substrates, following only a 2-h culture. Greatersubsequent functions leading to new bone synthesis has also been reported on nanofibrous com-pared to nano- and conventional spherical alumina [111]. Thus, perhaps not only is the nanometergrain size of components of bone important to mimic in materials, but the aspect ratio may also bekey to simulate in synthetic materials to optimize bone cell response.

Another class of novel biologically inspired nanofiber materials that have been investigated fororthopedic applications are self-assembled helical rosette nanotubes [113]. These organic compoundsare composed of guanine and cytosine DNA pairs that self-assemble when added to water to formunique nanostructures (Figure 22.15). These nanotubes have been reported to be 1.1 nm wide and upto several millimeters wide [113]. Compared to currently used titanium, recent studies have indicatedthat osteoblast function is increased on titanium coated with helical rosette nanotubes (Figure 22.15)[113]. Although in these studies it has not been possible to separate the influence of nanometerdimensions from the effects of nanotube chemistry on cell functions, it is clear that these nanotubesare another category of novel nanostructured materials that can be used to promote bone formation.It is also intriguing to consider what role self-assembled nanofibers may play in orthopedics sincebone itself is a self-assembled collection of nanofibers.

In this context it is important to mention that only nanophase materials can mimic the unique aspectratio of HA and proteins found in the extracellular matrix of bone; it is not possible for micron-sized


Group ofaluminananofibers

FIGURE 22.14 Transmission electron microscope image of alumina nanofibers. Compared to spherical con-ventional alumina, increased functions of osteoblasts have been reported on nanophase fibrous alumina. Scalebar � 10 nm. (From Price, R.L. et al., J. Biomed. Mater. Res., 67, 1284, 2003. With permission.)

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materials to simulate the unique nanometer constituent components of bone. As mentioned previously,results concerning the importance of nanofibrous materials in promoting functions of osteoblasts havebeen reported for carbon and polymer molds of carbon nanofibers (Figure 22.11) [101]. These findingsconsistently testify to the unprecedented ability to create nanomaterials to mimic the dimensions ofcomponents of physiological bone to promote new bone formation.

22.6 METAL NANOMATERIALS

Although much work has been conducted on nanophase ceramics for orthopedic applications todate, several recent studies have focused on the analysis of bone regeneration on nanophase metals.Metals investigated to date include titanium, Ti6Al4V, and CoCrMo [114]. While many haveattempted to create nanostructured surface features using chemical etchants (such as HNO3) on tita-nium, results concerning increased bone synthesis have been mixed [58]. Moreover, through the useof chemical etchants it is unclear what the cells may be responding to — changes in chemistry orchanges in topography. For this reason, as was done for the ceramics in this chapter, it is important


Cross-sectional view Longitudinal view(a)

Helicalrosettenanotubes

0 200 400 600

50 nm0

200

0.0 nm

nm

20.0 nm

10.0 nm

400

600

(b)

FIGURE 22.15 Helical rosette nanotubes. Drawing of the cross-sectional (left) and longitudinal (right) viewof self-assembled helical rosette nanotubes is depicted in (a) while helical rosette nanotubes coated on titaniumis depicted in (b). Note the nanophase dimension of these organic tubes. Increased osteoblast function has beenobserved on helical rosette nanotubes coated on Ti. (From Chun, A. et al., Nanotechnology, 15, S234, 2004.With permission.)

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to focus on studies that have attempted to minimize large differences in material chemistry andfocus only on creating surfaces that alter in their degree of nanometer roughness.

One such study by Ejiofor et al. [114] utilized traditional powder metallurgy techniques with-out the use of heat to avoid changes in chemistry to fabricate different particle size groups of Ti,Ti6Al4V, and CoCrMo (Figure 22.16). Increased osteoblast adhesion, proliferation, synthesis ofextracellular matrix proteins (like alkaline phosphatase and collagen), and deposition of calcium-containing mineral was observed on respective nanophase compared to conventional metals [114].This was the first study to demonstrate that the novel enhancements in bone regeneration previouslyseen in ceramics by decreasing grain size can be achieved in metals.

Interestingly, when Ejiofor et al. [114] examined spatial attachment of osteoblasts on the sur-faces of nanophase metals, they observed directed osteoblast attachment at metal grain boundaries(Figure 22.17). Because of this, the authors speculated that the increased osteoblast adhesion maybe due to more grain boundaries at the surface of nanophase compared to conventional metals. Aswas the case with nanophase ceramics [94,99], it is plausible that protein adsorption and


Nanophase Ti Conventional Ti

Nanophase Ti6Al4V Conventional Ti6Al4V

Nanophase CoCrMo Conventional CoCrMo

FIGURE 22.16 Scanning electron micrographs of nanophase metals. Increased functions of osteoblasts havebeen observed on nanophase compared to conventional c.p. Ti, Ti6Al4V, and CoCrMo. Scale bar = 1 µm fornanophase Ti/Ti6Al4V and 10 µm for conventional Ti/Ti6Al4V. Scale bar = 10 µm for nanophase and conven-tional CoCrMo. (From Ejiofor, J.U. and Webster, T.J., ASM Conference, Las Vegas, NV, 2004. With permission.)

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conformation at nanophase metal grain boundaries may be greatly altered compared to nongrainboundary areas and conventional grain boundaries; in this manner, protein interactions at grainboundaries may be key for osteoblast adhesion.

22.7 POLYMERIC NANOMATERIALS

For ceramics and metals, most studies conducted to date have created desirable nanometer surfacefeatures by decreasing the size of constituent components of the material, e.g., a grain, particle, orfiber. However, due to the versatility of polymers, many additional techniques exist to createnanometer surface roughness values. In addition, polymers contribute even further to rehabilitatingdamaged tissue by possibly providing a degradable scaffold that dissolves within a controllable timewhile the native tissue reforms. Techniques utilized to fabricate nanometer features on polymersinclude e-beam lithography, polymer de-mixing, chemical etching, cast-mold techniques, and theuse of spin-casting [114–122]. For those that have been applied to bone regeneration, chemicaletching followed by mold casting and polymer de-mixing techniques have received the most atten-tion [115,116].

For chemical etching techniques, polymers investigated to date include PLGA (Figure 22.18), PU,and polycaprolactone [116,118–120]. The idea proposed by Kay et al. has been to treat acidic poly-mers with basic solutions (i.e., NaOH) and basic polymers with acidic solutions (i.e., HNO3) to cre-ate nanosurface features [116]. Kay et al. observed greater osteoblast adhesion on PLGA treated withincreasing concentrations and exposure times of NaOH only on two-dimensional films. As expected,data were also provided indicating larger degrees of nanometer surface roughness with increased con-centrations and exposure times of NaOH on PLGA. Park et al. [118] took this one step further andfabricated three-dimensional tissue engineering scaffolds by NaOH treatment of PLGA. When com-paring osteoblast functions on such scaffolds, even though similar porosity properties existed betweennontreated and NaOH-treated PLGA (since similar amounts and sizes of NaCl crystals were used tocreate the pores through salt-leaching techniques), greater numbers of osteoblasts were counted on andin NaOH-treated PLGA [118]. Unfortunately, due to these fabrication techniques, it is unclear whetherthe altered PLGA chemistry or nano-etched surface promoted osteoblast adhesion; however, in light


Osteoblasts



FIGURE 22.17 Scanning electron micrographs of adherent osteoblasts on nanophase c.p. Ti. Directedosteoblast adhesion on nanophase metal grain boundaries has been reported. Scale bar = 100 µm for top and10 µm for bottom. Adhesion time = 30 min. (From Ejiofor, J.U. and Webster, T.J., ASM Conference, LasVegas, NV, 2004. With permission.)

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of the previous studies mentioned in this chapter, the authors of Ref. [118] suggested that the nanome-ter surface roughness of the NaOH-treated PLGA played an important role [118].

Studies have also been conducted on cell responses to polymers with changes in nanometer sur-face roughness without changes in chemistry. Specifically, Li et al. utilized polymer de-mixingtechniques to create well-controlled nanometer islands of polystyrene and polybromo-styrene[121]. Although osteoblast functions have not been tested on these constructs to date, fibroblastmorphology was significantly influenced by incremental nanometer changes in polymer islanddimensions (Figure 22.19). Again, this study points to the unprecedented control that can be gainedover cell functions by synthesizing materials with nanometer surface features.

Although not related to orthopedic applications, vascular and bladder cell responses have alsobeen promoted by altering the topography of polymeric materials in the nanometer regime[116,118,120,122]. In these studies, chondrocytes [116], bladder [120], and vascular smooth mus-cle cell [119] adhesion and proliferation were greater on two-dimensional nanometer surfaces ofbiodegradable polymers such as PLGA, PU, and polycaprolactone; similar trends have recentlybeen reported on three-dimensional PLGA scaffolds [122].

22.8 COMPOSITE NANOMATERIALS

Owing to the previous information of increased osteoblast function on ceramics [100] and polymers[111], bone cell function on nanophase ceramic polymer composites have also been determined.Specifically, studies conducted to date show promoted osteoblast responses on composites of PLGAcombined separately with nanophase alumina, titania, and HA (30:70 wt% PLGA/ceramic)(Figure 22.20) [123]. For example, up to three times more osteoblasts adhered to PLGA when itcontained nanophase compared to conventional titania particles [116]. Since similar porosity


(b) Nano-structured PLGA (a) Conventional PLGA

(c) Conventional PLGA (d) Nano-structured PLGA

FIGURE 22.18 Scanning electron micrographs of conventional and nanophase PLGA scaffolds. Increasedosteoblast functions have been demonstrated on nanophase PLGA scaffolds. Scale bar = 10 µm. (From Park,G.E. et al., Biomaterials, 26, 3075–3082, 2005. With permission.)

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(both percentages and diameters) existed between PLGA with conventional titania compared tonanophase titania, another novel property of nanophase ceramic composites was elucidated in thisstudy: increased osteoblast functions. This is in addition to numerous reports in the literature high-lighting greater toughness of nanophase compared to conventional ceramic/polymer composites[44–46].

Moreover, promoted responses of osteoblasts have also been reported when carbon nanofibers wereincorporated into polymer composites; specifically, three times the number of osteoblasts adhered onPU with increasing weight percentages of nanometer carbon fibers when compared with conventionaldimension carbon fibers (Figure 22.21) [124]. As mentioned, reports in the literature have demonstratedhigher osteoblast adhesion on nanophase carbon fibers in comparison with conventional carbon fibers(or titanium [ASTM F-67, Grade 2] [124]), but this study demonstrated greater osteoblast adhesion withonly a 2 wt% increase of carbon nanofibers in the PU matrix. Up to three and four times the number ofosteoblasts that adhered on the 100:0 PU/CN wt%, adhered on the 90:10 and the 75:25 PU/CN wt%composites, respectively [124]. This exemplifies the unprecedented ability of nanophase materials toincrease functions of bone cells whether used alone or in polymer composite form.


Polymerdemixednanoislands

Fibroblastfilopodia

(a) (b)

(c) (d)

FIGURE 22.19 Polymer nanoislands created by de-mixing polystyrene and polybromo-styrene. Altered cellfunctions have been observed on polymer nanoislands compared to conventional polymer topographies. (a)through (d) represents increased magnification. (From Dalby, M.J. et al., Biomaterials, 23, 2945, 2002. Withpermission.)

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22.9 AREAS OF APPLICATION

While there has already been some effort on incorporating nanotechnology into orthopedic appli-cations, it is clear that this is only the beginning for the incorporation of nanotechnology intobiology. In the following sections some additional avenues are highlighted.

22.9.1 DRUG DELIVERY

Polymers have found a significant role in the development of novel drug delivery systems.Biomaterials for muco-adhesive drug delivery applications have been improved through theaddition of PEG as an adhesion promoter [126–129]. Additionally, smart hydrogel drug carriershave been molecularly designed to carry safely proteins and peptides to the duodenum region,avoiding the harsh, acidic conditions of the stomach and the proteolytic enzymes present along thegastrointestinal tract [130–132]. Drug delivery systems composed of degradable polymers, such as


ConventionalTiO2

PLGA: Conventional TiO2(70:30 wt%)

NanophaseTiO2

PLGA: Nanophase TiO2

(70:30 wt%)

FIGURE 22.20 Scanning electron micrographs of poly-lactic-glycolic acid (PLGA): titania composites.Increased osteoblast function has been observed on polymer composites containing nanophase compared toconventional ceramics. Scale bar = 10 µm. (From Webster, T.J. and Smith, T.A., J. Biomed. Mater. Res., 74,677–686, 2004. With permission.)

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polyanhydrides [133–136], polyorthoesters [137,138], and PLGA [139,140], have been imple-mented in chemically controlled drug delivery.

Lu and Chen [141] have highlighted recent developments in the nanofabrication of biodegrad-able drug delivery systems, and Leoni and Desai [142] have offered a review of the abilities to cre-ate nanoporous structures containing an optimal pore size and distribution. Nanoporous structures[143] have the ability to allow mass transport of desirable compounds but limit those that are unde-sirable. An example given in the aforementioned review is the nanoporous encapsulation of pan-creatic islet cells. The controlled pore size could allow certain molecules such as glucose transportbut significantly hinder the diffusion of molecules responsible for causing immune response-medi-ated device failure such as immunoglobulin G and M molecules (IgG and IgM).

Nanodrug delivery systems are quickly evolving in their ability to integrate biologically com-plex components into a functional nanodevice. Lee et al. [144] offer the bacmid process as anobject-oriented approach for designing novel nanosystems and outline the possibility of using thistype of design process for effective delivery of vaccines. Santini et al. [40] have demonstrated theability to deliver nanoliter quantities of therapeutics on demand from an array of sealed reservoirs.Drug discovery and delivery are becoming sciences that encompass skills in nanotechnology,


(a) 100:0 (PU:CN wt%) (b) 98:2 (PU:CN wt%)

(c) 90:10 (PU:CN wt%) (d) 75:25 (PU:CN wt%)

(e) 0:100 (PU:CN wt%)

FIGURE 22.21 Scanning electron micrographs of poly-ether-urethane (PU): carbon nanofibers (wt.%) com-posites. Increased functions of osteoblasts have been observed on polymer composites containing carbonnanofibers. Scale bar = 1 µm. (From Price, R.L. et al., Biomaterials, 24, 1877, 2003. With permission.)

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microtechnology, and biology to design systems effectively more capable of achieving efficient andeffective therapeutic treatments [145].

Much effort has been dedicated to engineering nanoparticulate drug delivery systems [146,147].Surface modification allows the specific targeting of particles and enhances their ability to interactwith certain types of cells [148]. Size plays a key role in the ability of particles to participate inintracellular uptake, and biodegradable nanoparticles can be used as sustained-release delivery sys-tems once inside the cytoplasm [149].

22.9.2 TISSUE ENGINEERING

Tissue engineering strives to create living tissue and organs through the use of synthetic, hybrid, ornatural materials that have been designed or fabricated in a way to elicit a desirable cellularresponse from the scaffold [150]. The major thrust of developing materials for tissue engineering isproviding the cells an environment in which they can continue their normal functionality.Biodegradable and resorbable materials are favorable due to the lack of necessity of a materialstructure to be present once the matrix has been formed. Poly(lactic acid), poly(glycolic acid), andtheir copolymers (PLGA) were first utilized in the development of biodegradable materials for tis-sue engineering applications, followed by other types of materials that include PUs [151], polyan-hydrides [152,153], and, more recently, poly(ether anhydrides) [154].

This chapter has provided some nanotechnology-based examples of tissue engineeringadvances in orthopedics. In addition, Tsang and Bhatia [155] offer an extensive review into the fab-rication techniques developed to aid in the development of novel tissue scaffolds. Significantadvances in fabrication using heat, light, adhesives, and molding are elaborated upon. The use ofcells to self-assemble native cellular matrices and cell/scaffold hybrids are also highlighted.Biomimetic materials are actively being pursued as integral components of novel tissue engineer-ing biomaterials [156,157]. Various techniques are being employed to take advantage of the abilityto modify the surface of tissue engineering materials, with RGD modification receiving the mostattention [158].

Synthetic hydrogels have traditionally been employed in the development of tissue engineeringscaffolds due to their high biocompatibility, hydrophilicity, and tissue-like properties. The molecu-lar design of these materials affords the engineer the ability to impart certain physical propertiesinto the device to obtain the necessary physiological response. Poly(lactic acid)-g-poly(vinylalchol) (PLA-g-PVA) have been developed for heart valves [159], and PVA/poly(vinyl pyrrolidone)(PVA/PVP) blends have been proposed for nucleus pulposus replacement [160]. Natural materialsthat have been developed for tissue engineering applications include collagen, hyaluronic acid, algi-nate, and chitosan [161].

Hybrid materials are being utilized to take advantage of the ability to synthesize polymericmaterials with specific properties combined with a bioactive entity that helps elicit a particular bio-logical response. Chondroitin sulfate, a biological polymer and PVA, a synthetic polymer, havebeen used to synthesize hydrogels that promote chondrogenesis [162]. Mixtures of peptides andsynthetic polymers are combined in order to mimic extracellular matrix proteins to enable naturalwound healing and reduce the formation of fibrous encapsulation. RGD has been used frequentlyto impart these properties into a biomaterial [158].

22.9.3 BIOLOGICAL MICRO-ELECTRO-MECHANICAL SYSTEMS

Most research focusing on biological micro-electro-mechanical systems (BioMEMS) is for theiruse in diagnostic devices and for the detection of DNA, viruses, proteins, and other biologicallyderived molecules [163]. Nanoscale BioMEMS could allow for the real-time detection and analy-sis of signaling pathways, which would further our knowledge and understanding of the basicmechanisms and functions of the cell. While nanoscale BioMEMS is at its infancy, it is clear thatnanotechnology will play an important role in its development.


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22.10 CONSIDERATIONS AND FUTURE DIRECTIONS

Although preliminary attempts to incorporate nanotechnology into biomedical applications seempromising, numerous urgent questions still remain with regard to this new field. First and foremost,the question of safety of nanoparticles once in the human body remains largely unanswered bothfrom a manufacturing point of view and when used in full or as a components of an implantabledevice. Since such particles are smaller than many pores of biological tissues, it is clear that thisinformation will have to be obtained before further consideration of implantable nanomaterials isundertaken. Such nanoparticles can easily become dislodged from implants during surgical implan-tation or by fragmentation of articulating components of a joint prosthetic composed of nanophasematerials. Although preliminary in vitro studies highlight a less adverse influence of nanometercompared to micron particulate wear debris on bone cell viability [164,165], many more experi-ments are needed, especially in vivo to evaluate their efficacy.

Specifically for orthopedic applications, additional questions remain. For example, once exactoptimal nanometer surface features are elucidated for increasing bone regeneration, inexpensivetools that can be used in industry will be required. In this context, if the only nanofabricationdevices that can be used to synthesize desirable nanometer surface features for bone regenerationare e-beam lithography or other equally expensive techniques, industry may not participate in thisboom of nanotechnology at the intersection of tissue engineering. Inexpensive but effectivenanometer synthesis techniques must continually be a focus of many investigators.

The direction of the nanotechnology should be and is geared toward dealing with these issues.For example, according to the U.S. government’s research agenda, current and future broad inter-ests in nanobiomedical activity can be categorized into three broad related fronts [51,52]:

1. development of pharmaceuticals for inside-the-body applications — such as drugs foranti-cancer and gene therapy

2. development of diagnostic sensors and lab-on-a-chip techniques for outside-the-bodyapplications — such as biosensors to identify bacteriological infections in biowarfare

3. development of prostheses and implants for inside-the-body uses

Whereas the European governments emphasize commercial applications in all the three fronts men-tioned above, according to Marsch [52], the U.S. government tends to lean toward fundamentalresearch on biomedical implants and biodefense, leaving commercial applications to industry. Bothclassifications identify nanophase biomedical implants as a potential interest. The biological andbiomimetic nanostructures to be used as orthopedic implants involve some sort of an assembly inwhich smaller materials later on assume the shape of a body part, such as hip bone. These final bio-mimetic, bulk nanostructures can start with a predefined nanochemical (like an array of large reac-tive molecules attached to a surface) or nanophysical (like a small crystal) structure. It is believedthat by using these fundamental nanostructured building blocks as seed molecules or crystals, alarger bulk material will self-assemble or keep growing by itself.

In summary, it is now believed that significant evidence exists that highlights the promise nan-otechnology has for biological applications, particularly in the bone arena. Clearly, nanomaterialsas mentioned here are at their infancy and require much more testing before their full potential canbe realized. However, even if nanophase materials never make it to the marketplace due to safetyconcerns, we have already learned much about how cells interact with surfaces through their appli-cation in the orthopedic environment.

ACKNOWLEDGMENTS

JBT is supported through a Department of Homeland Security graduate research fellowship. JBTand NAP thank NIH and NSF for support of the research summarized here. MS and TJW thank theNSF and NIH for funding part of the research summarized here through the Bio-NanotechnologyNational Initiative.


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